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. 2019 May:531:141-148.
doi: 10.1016/j.virol.2019.03.006. Epub 2019 Mar 9.

A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43

Florence Maurier  1 , Delphine Beury  1 , Léa Fléchon  2 , Jean-Stéphane Varré  2 , Hélène Touzet  2 , Anne Goffard  1 , David Hot  1 , Ségolène Caboche  3
Affiliations

A complete protocol for whole-genome sequencing of virus from clinical samples: Application to coronavirus OC43

Florence Maurier et al. Virology. 2019 May.

Abstract

Genome sequencing of virus has become a useful tool for better understanding of virus pathogenicity and epidemiological surveillance. Obtaining virus genome sequence directly from clinical samples is still a challenging task due to the low load of virus genetic material compared to the host DNA, and to the difficulty to get an accurate genome assembly. Here we introduce a complete sequencing and analyzing protocol called V-ASAP for Virus Amplicon Sequencing Assembly Pipeline. Our protocol is able to generate the viral dominant genome sequence starting from clinical samples. It is based on a multiplex PCR amplicon sequencing coupled with a reference-free analytical pipeline. This protocol was applied to 11 clinical samples infected with coronavirus OC43 (HcoV-OC43), and led to seven complete and two nearly complete genome assemblies. The protocol introduced here is shown to be robust, to produce a reliable sequence, and could be applied to other virus.

Keywords: Bioinformatics; Complete genome; Coronavirus; High-throughput sequencing.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Schematic representation of the complete protocol for viral sequencing directly from clinical samples.
Fig. 2
Fig. 2
Difference observed between sequences generated from alignment combined with consensus extraction and full-amplicon based extraction.
Fig. 3
Fig. 3
Coronavirus genome amplification trials by RT-PCR for different fragment lengths. A: Capillary electrophoresis profiles of RT-PCR products of the obtained different fragments. B: Agarose gel-like profiles. L: ladder with the sizes alongside (in bp); a to j: RT-PCR product from ~500p to ~5000 bp with an increment-step of ~500 bp.
Fig. 4
Fig. 4
Study of the cluster size distribution for the 99 amplicons (PCR01 to PCR99) from the MDS4 sample. The top panel shows the number of reads for each amplicon (in black) and the number of reads in the biggest cluster (in gray). The bottom panel shows the proportion of each clusters for each amplicon. The proportion of the biggest cluster is in black and the proportion of clusters containing less than 1% of reads were summed up and are in gray.
See this image and copyright information in PMC

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